This invention relates to plasmids for viruses, methods for engineering the plasmids and methods for analyzing virus replication.
The bovine viral diarrhea virus, or BVDV, is the prototype species in the Pestivirus genus of the Flaviviridae. Positive strand RNA viruses, such as the poliovirus and the BVDV, have a genome consisting of a single molecule of RNA. Pure RNA extracted from either highly concentrated and purified poliovirus or BVDV can be transfected into a susceptible cell, which results in the production of infectious progeny virus by the cell. A homogeneous preparation of viral RNA molecules is infectious upon transfection.
The usefulness of RNA molecules from BVDV and poliovirus is limited because they are many thousand nucleotides in length and cannot be engineered in vitro to change their sequence using current technology. By contrast, DNA molecules of similar length, cloned into plasmids, can be manipulated with restriction enzymes and synthetic oligodeoxynucleotides to obtain any desired sequence.
It is known that the entire RNA sequence of some viruses can be cloned into a plasmid. Such plasmids can be used to produce RNA molecules in vitro that mimic the properties of the RNA extracted from the purified virus. The RNA synthesized in vitro using a plasmid DNA as a template produces the virus upon transfection into susceptible cells. Plasmids with this property are known as "infectious clones."
BVDV remains enzootic in cattle populations throughout the world. Current vaccines are either relatively unsafe or ineffective in control and eradication programs. Efficient reverse genetics approaches using an infectious molecular clone of the BVDV genome may contribute to the development of rationally-designed safe and efficacious vaccines.
The construction of an infectious molecular clone of BVDV, its subsequent modification to introduce a sequence tagged site and the production of a chimeric virus expressing a surface glycoprotein from a different strain, manipulating the BVDV genome in vitro and its potential effectiveness for reverse genetic analyses of pestivirus are described hereinafter.